JP2016013981A - Tubulin polymerization activity regulator, food and agent comprising the same, and method of producing tubulin polymerization activity regulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tubulin polymerization activity regulator derived from Ganoderma lingzhi extract, foods, cosmetics and pharmaceuticals using the same, and a method of producing the tubulin polymerization activity regulator.SOLUTION: A tubulin polymerization activity regulator comprises a component extracted from Ganoderma lingzhi with solvent comprising water or organic solvent as an active ingredient, and has the effect of promoting or inhibiting tubulin polymerization. There is also provided foods, cosmetics and pharmaceuticals comprising the tubulin polymerization activity regulator.

Description

The present invention relates to a tubulin polymerization activity regulator containing an extract of Ganoderma lingzhi , particularly its fruiting body, a food and a drug containing the tubulin polymerization activity regulator, and a method for producing a tubulin polymerization activity regulator. About.

  Tubulin is a protein in eukaryotic cells that forms microtubules and centrosomes. During cell mitosis, tubulin is polymerized and extended from each centrosome of the intracellular bipolar to the central pair of chromosomes in the dividing cell. As a result of depolymerization of phosphorus, the chromosomes migrate to both cell poles and are distributed to each dividing cell. This tubulin is composed of a heterodimer composed of α-tubulin and β-tubulin (Non-patent Document 1). Of these, it is important for cell division to maintain a good balance between tubulin polymerization and depolymerization. If this balance is compromised, normal cells and tumor cells will not divide correctly and cannot proliferate. .

  Taxol (TAXOL, a registered trademark of Bristol-Myers Squibb, Inc., commonly known as an antitumor agent. Generic name "Paclitaxel") promotes polymerization of tubulin and polymerizes in bundles of microtubules. By stabilizing this state, the depolymerization of microtubules is inhibited, and as a result, the growth of tumor cells is suppressed. On the other hand, vinblastine is thought to suppress tumor cell growth by binding to β-tubulin, arresting the cell cycle at metaphase, and inhibiting tubulin polymerization ( Non-patent document 2).

  If a substance that can promote or inhibit tubulin polymerization (a regulator of tubulin polymerization activity) is newly found, can suppress the division of cells such as tumors, and induce apoptosis, tubulin polymerization activity It is also useful from the viewpoint of contributing to the elucidation of the regulation mechanism and the discovery of new antitumor agents (Non-patent Document 3). Therefore, various tubulin polymerization activity regulators are desired.

  The present inventors discovered "ganoderic acid DM (ganoderic acid DM)" which has a high effect of regulating (that is, promoting or inhibiting) the polymerization of tubulin in the same manner as taxol (paclitaxel) and vinblastine described above. Have already been reported (Patent Document 1).

JP2013-043874A

Downing, K. H., Nogales, E., Tubulin structure: insights into microtubule properties and functions, Current Opinion in Structural Biology, 8 (6): 785-791 (1998) Checchi, P. M., nettles, J. H., Zhou, J., Snyder, J. P, Joshi, H. C., Microtubule-interacting drugs for cancer treatment, 24 (7) 361-365 (2003) Jordan A1, Hadfield JA, Lawrence NJ, McGown AT., “Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle.” [Online], July 1998, Medicinal Research Reviews, [2014 June 11 search], Internet <URL: http://www.ncbi.nlm.nih.gov/pubmed/9664292>

  The inventors of the present invention have been diligently investigating the tubulin polymerization activity and the like of trippenoids contained in extracts and extracts extracted by different methods from ganoderma (particularly fruit bodies) by various extraction methods. Depending on the extraction method, the main components contained in the extract and their composition ratio may be different. Depending on the resulting extract, tubulin polymerization may be accelerated (or inhibition of tubulin depolymerization) Conversely, it has been found that polymerization of tubulin may be suppressed. Furthermore, when other reishi tripenoids other than the conventionally known ganodermic acid DM contained in the extract of ganoderma were also examined, the ganoderma extract promoted tubulin polymerization more than ganodermic acid DM. The present inventors have found that there are a large number of tripenoids having such activity, and have completed the present invention.

  Accordingly, the present invention is derived from the above extract of ganoderma tubulin polymerization, which can increase tubulin polymerization activity (accelerate polymerization) or decrease tubulin polymerization activity (suppression polymerization, inhibit polymerization). It is an object of the present invention to provide an activity regulator, a food, a cosmetic and a pharmaceutical using the same, and a method for producing the tubulin polymerization activity regulator. Furthermore, for example, an object of the present invention is to provide a research reagent that contributes to the identification of a chemical structure necessary for efficient regulation of tubulin polymerization activity.

The present invention provides the following tubulin polymerization activity regulators and the like.
[1] Tubulin characterized by containing as an active ingredient a component obtained by extraction from Ganoderma lingzhi with a solvent containing water (aqueous solvent) and having an action of inhibiting tubulin polymerization. Polymerization activity regulator.

[2] It is characterized in that it contains tripenoids obtained by extraction from Ganoderma lingzhi with a solvent containing an organic solvent (organic solvent) as an active ingredient and has an action of promoting tubulin polymerization. Tubulin polymerization activity regulator.

[3] Among the following (1) to (34), the tripenoid is one or more selected from the group consisting of (1) to (6) and (9) to (33) The tubulin polymerization activity regulator as set forth in [2], characterized in that it is a compound (note that the numbers (1) to (34) are given for the convenience of explanation of the present invention).
(1) Ganoderic acid Z (Ganoderic acid Z),
(2) Ganoderic acid N (Ganoderic acid N),
(3) Ganoderic acid S (Ganoderic acid S),
(4) Ganoderic acid F,
(5) Ganoderic acid SZ (Ganoderic acid SZ),
(6) Ganoderic acid LM2 (Ganoderic acid LM2),
(7) Ganoderic acid DM (Ganoderic acid DM),
(8) Ganoderic acid A,
(9) Ganoderic acid AM1 (Ganoderic acid AM1),
(10) Ganoderic acid B (Ganoderic acid B),
(11) Ganoderic acid D (Ganoderic acid D),
(12) Ganoderic acid Y,
(13) Ganoderic acid TN (Ganoderic acid TN),
(14) Ganoderic acid TQ (Ganoderic acid TQ),
(15) Ganolucidic acid A,
(16) Ganodermanontriol,
(17) Ganodermanondiol,
(18) Ganoderol A (Ganoderol A),
(19) Ganoderol B,
(20) Ganoderiol F,
(21) Lucialdehyde A,
(22) Lucialdehyde B,
(23) Ganoderic acid C1 (Ganoderic acid C1),
(24) Ganoderic acid C2 (Ganoderic acid C2),
(25) Ganoderic acid C6,
(26) Ganoderic acid H,
(27) Ganoderic acid K,
(28) Ganoderic acid TR (Ganoderic acid TR),
(29) Ganoderenic acid A,
(30) Ganoderenic acid C,
(31) Ganoderenic acid D,
(32) Ganoderenic acid F,
(33) Ganoderenic acid H,
(34) Ganoderic acid DM methyl ester

  [4] The tubulin polymerization activity regulator according to [1], wherein the active ingredient is a polysaccharide containing β-glucan.

  [5] The tube according to any one of [1] to [3], wherein the solvent is water when it is an aqueous solvent, and is chloroform, alcohol, or hydrous alcohol when it is an organic solvent. Phosphorus polymerization activity regulator.

  [6] Food (health supplement), cosmetic for preventing or reducing diseases related to tubulin polymerization, comprising the tubulin polymerization activity regulator according to any one of [1] to [5] as an active ingredient Fee or medicine.

  [7] A research reagent containing the tubulin polymerization activity regulator according to any one of [1] to [5] as an active ingredient.

  [8] The food and cosmetic according to [6], wherein the disease is prevention of disease such as neoplastic disease, psoriasis, stenosis, myocardial infarction, glomerulonephritis, improvement of symptoms, or transplant rejection Or pharmaceuticals.

  [9] A fungicide, an anthelmintic or a herbicide containing the tubulin polymerization activity regulator according to any of [1] to [5] as an active ingredient.

  [10] A bittering agent comprising the tubulin polymerization activity regulator according to any one of [1] to [5] as an active ingredient.

[11] A method of polymerizing tubulin, comprising a step of extracting a component having an inhibitory effect on tubulin polymerization from a ganoderma lingzhi using a solvent containing water (aqueous solvent). A method for producing a tubulin polymerization activity regulator having an inhibitory action.

[12] Tubulin polymerization characterized by including a step of extracting a component having a tubulin polymerization promoting action from Ganoderma lingzhi using a solvent (organic solvent) containing an organic solvent. A method for producing a tubulin polymerization activity regulator having an action of promoting aging.

  According to the present invention, a tubulin polymerization activity regulator derived from Ganoderma can be regulated by promoting or inhibiting the polymerization of tubulin, a food and a drug containing the same, and an efficient tubulin polymerization activity regulator. A manufacturing method is provided.

FIG. 1 is a view showing a binding state of ganoderic acid TQ (βnoderic acid TQ) and β-tubulin (β-Tubulin) having high activity for promoting tubulin polymerization. This binding state is obtained by using the binding structure of paclitaxel (Taxol) and tubulin (PDB code 1JFF) with the software “Molgro Virtual Docker” (Northern Science Consulting) to replace ganoderic acid TQ (instead of paclitaxel). It was obtained from the result of docking simulation of the bond between this ganoderic acid TQ and β-tubulin (β-Tubulin) using Ganoderic acid TQ) (the same applies to FIGS. 2 to 4 below). In FIG. 1, a part of amino acid residues (Thr276 (β), Arg284 (β) of β-tubulin (the whole molecule is not shown) is located at the acetoxy group (—OAc) site of Ganodermic Acid TQ as a ligand. )) Is bound. In FIG. 1, a part of the amino acid residues (Thr276 (β), Arg284 (β)) of β-tubulin involved in the binding are indicated by rectangles. According to FIG. 1, the chemical structure of a highly active tubulin polymerization accelerator that promotes the polymerization of tubulin and the binding mechanism between the tubulin polymerization accelerator and β-tubulin can be grasped. FIG. 2 is a diagram showing the binding mode between ganodermic acid TN (ligandic acid TN) as a ligand and β-tubulin. As in the case of FIG. 1, some of the amino acid residues (His229 (β), Gly370 (β), Thr276 (β)) of β-tubulin involved in binding (the whole molecule is not shown) are rectangular. It is shown. From FIG. 2, ganodermic acid TN contributes to the elucidation of the chemical structure contributing to the promotion of tubulin polymerization and the binding mode of β-tubulin and the ligand binding to β-tubulin in this case. I can say that. FIG. 3 is a diagram showing the binding mode of Ganoderic acid S and β-tubulin. From the comparison of FIG. 3 with FIGS. 1 and 2, the chemical structure of the compound affecting the activity of promoting the polymerization of tubulin in a binding mode different from that of ganoderic acid TQ, S, and in this case The binding mode between β-tubulin and the ligand that binds to β-tubulin can be grasped. FIG. 4 is a diagram showing a binding mode between paclitaxel (taxol) and β-tubulin. FIG. 5 shows the tubulin polymerization activity (ΔRFU average) examined for the compounds (1) to (34) derived from Ganoderma lingzhi and the interaction between each compound and β-tubulin examined by the docking simulation. The correlation between the E-Total value that is the sum of the energy (E1) and the internal energy (E2) of each compound is shown. In FIG. 5, the symbols (1), (3) to (8), (10), (12) to (14), (16) to (24), (28), and (34) represent the above compounds ( 1), (3) to (8), (10), (12) to (14), (16) to (24), (28), and (34) are shown. R ² represents a correlation coefficient, and y represents a linear approximation formula. ΔRFU average indicates tubulin polymerization activity. FIG. 5 shows a correlation between tubulin polymerization activity (ΔRFU average) and E-Total value. FIG. 6 shows a chloroform extract of Ganoderma lingzhi (30 minutes at room temperature using chloroform, shaking extraction: Test Example 4), and a hot water extract (121 ° C., 20 minutes autoclave extraction: Test Example 4). Is a graph showing the change over time in the amount of tubulin polymer (RFU) when evaluated using a “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.) at a predetermined concentration. For comparison, the case where Paclitaxel (Paclitaxel) 2.73 μM (2.33 μg / mL), Vinblastine 2.73 μM (2.48 μg / mL) and control (no addition) are also shown. According to FIG. 6, the amount of tubulin polymer (RFU) is the time (0 to 60 minutes) even without any treatment (control (no addition): the third solid line from the top in FIG. 6). It can be seen that it increases gradually. On the other hand, when treated with the chloroform extract (273 μg / mL: dotted line) according to the present invention, compared with the case of treatment with the comparative control paclitaxel (2.33 μg / mL: first solid line from the top of FIG. 6), Although the degree of increase in the amount of tubulin polymer (RFU) over time (tubulin polymerization activity: ΔRFU average) is low, it shows an increasing tendency as compared with the control (no addition). In addition, according to FIG. 6, in the hot water extract according to the present invention (1820 μg / mL: two-dot chain line), the degree of increase in the amount of tubulin polymer (RFU) with time is compared with the comparison vinblastine ( 2.48 μg / mL: slightly higher than the first broken line (from the bottom of FIG. 6), but, like vinblastine, there is virtually no increase in the amount of tubulin polymer (RFU) over time, and control (no It can be said that the amount of tubulin polymer (RFU) does not increase compared to (addition). Therefore, according to FIG. 6, the extract extracted from Ganoderma lucidum with an organic solvent such as chloroform (ganoderma organic solvent extract) shows how the amount of tubulin polymer (RFU) changes. Antitumor effects and the like due to a mechanism similar to paclitaxel can be expected. Moreover, the hot water extract of Ganoderma can be expected to have an antitumor effect or the like by a mechanism similar to vinblastine. FIG. 7A shows the amount of tubulin polymer (RFU) when a tripenoid compound (18.2 μM) obtained from Ganoderma lingzhi was evaluated using “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). It is the graph which showed change with time. (A) Ganodermic Acid TQ, (B) Ganodermic Acid SZ, (C) Ganodermic Acid S, (D) Ganodermic Acid TN, (E) Ganodermanon Triol, (F) Ganodermic Acid AM1, (G) Ganodermanon triol, (H) Ganodermic acid D. For comparison, a case where Paclitaxel (Paclitaxel) 3 μM (2.6 μg / mL), Vinblastine 3 μM (2.7 μg / mL), and control (no addition) are also shown. FIG. 7B shows the amount of tubulin polymer (RFU) when a tripenoid compound (18.2 μM) obtained from Ganoderma lingzhi was evaluated using the “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). It is the graph which showed change with time. (A) Ganoderiolic F, (B) Ganodermic Acid TR, (C) Ganodermanone Triol, (D) Lucidaldehyde A, (E) Ganoderic acid A, (F) Ganoderic acid D, (G) Ganoderic acid F, (H) Ganodermic acid C2. For comparison, a case where Paclitaxel (Paclitaxel) 3 μM (2.6 μg / mL), Vinblastine 3 μM (2.7 μg / mL), and control (no addition) are also shown. FIG. 7C shows the amount of tubulin polymer (RFU) when a tripenoid compound (18.2 μM) obtained from Ganoderma lingzhi was evaluated using “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). It is the graph which showed change with time. (A) Ganodermic acid C6, (B) Ganodermic acid Y, (C) Ganodermic acid D, (D) Ganoderic acid K, (E) Ganoderic acid F, (F) Ganodermic acid H, (G ) Ganoderic acid C, (H) ganoderic acid C1. For comparison, a case where Paclitaxel (Paclitaxel) 3 μM (2.6 μg / mL), Vinblastine 3 μM (2.7 μg / mL), and control (no addition) are also shown. FIG. 7D shows the amount of tubulin polymer (RFU) when a tripenoid compound (18.2 μM) obtained from Ganoderma lingzhi was evaluated using the “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). It is the graph which showed change with time. (A) Ganodermic Acid Z, (B) Ganodermic Acid LM2, (C) Ganodic Acid A, (D) Ganodermic Acid DM, (E) Ganodermic Acid A, (F) Ganoderol B, (G) Ganoderol A, (H) Lucidaldehyde B. For comparison, a case where Paclitaxel (Paclitaxel) 3 μM (2.6 μg / mL), Vinblastine 3 μM (2.7 μg / mL), and control (no addition) are also shown. FIG. 7E shows the amount of tubulin polymer (RFU) when a tripenoid compound (18.2 μM) obtained from Ganoderma lingzhi was evaluated using “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). It is the graph which showed change with time. (A) Ganodermic acid B, (B) Ganodermic acid N. For comparison, a case where Paclitaxel (Paclitaxel) 3 μM (2.6 μg / mL), Vinblastine 3 μM (2.7 μg / mL), and control (no addition) are also shown. According to FIG. 7A to FIG. 7E, from the comparison of control (no addition), paclitaxel or vinblastine with each tripenoid compound, each triterpenoid obtained from ganoderma is tubulin from a point in time after paclitaxel acts. The amount of polymer (RFU) increases and each triterpenoid is thought to promote tubulin polymerization by a mechanism different from that of paclitaxel, and it can be seen that there is a possibility that it can be used together with paclitaxel. As a result, a synergistic effect of such combined use can be expected. In addition, when FIG. 8 to FIG. 9 are compared with FIG. 7, the tripenoids derived from Ganoderma are smaller in amount than the chloroform extract, but have higher tubulin polymerization activity (ΔRFU average) than the chloroform extract. It can be seen that Ganoderma derived triterpenoids are more effective than chloroform extracts as tubulin polymerization accelerators. FIG. 8 is a diagram showing the MS and MS / MS spectra of Ganoderic acid A (retention time 19.66 minutes). Measurement conditions: ("SHISEIDO" CAPCELL PACK C18 (column inner diameter: 1 mm, column length: 150 mm, average particle diameter of each packing material particle: 5 µm), injection amount: 5 µL, flow rate: 0.1 mL / min, LC (Detection wavelength: 252 nm, column temperature: 30 ° C.) Unless otherwise stated, the following MS and MS / MS spectra are the same. FIG. 9 is a diagram showing the MS and MS / MS spectrum of Ganoderic acid B (retention time 14.94 minutes). FIG. 10 is a diagram showing MS and MS / MS spectra of Ganolucidic acid A (retention time 8.12 minutes). FIG. 11 is a diagram showing the MS and MS / MS spectra of Ganoderic acid DM (retention time 67.47 minutes). FIG. 12 shows the MS and MS / MS spectra of Ganodermanontriol (retention time 62.23 minutes). FIG. 13 is a diagram showing MS and MS / MS spectra of ganoderic acid C1 (retention time 20.98 minutes). FIG. 14 is a diagram showing an MS and an MS / MS spectrum of Ganoderic acid TQ (retention time 81.14 minutes). FIG. 15 shows the MS and MS / MS spectra of Ganoderol A (retention time 98.69 minutes). FIG. 16 shows the MS and MS / MS spectra of Ganoderiol F (retention time 74.00 minutes). FIG. 17 is a diagram showing a chromatograph of a chloroform extract of Ganoderma lingzhi . Measurement conditions: (gradient); Line A: 0.1% AcOH-Water, Line B: 0,1% AcOH-AcCN, 25-35% B in 0-35 min, 35-45% B in 35-45 min, 45 -100% in 45-145 min, 100% B in 145-160 min Flow rate is 0.1 mm / min. 18 is a diagram showing the MS and MS / MS spectrum of peak 1 (retention time 15.00 minutes) of the chromatograph of FIG. FIG. 19 shows the MS and MS / MS spectrum of peak 2 (retention time: 15.00 minutes) in the chromatograph of FIG. FIG. 20 shows the MS and MS / MS spectra of peak 3 (retention time 66.22 minutes) of the chromatograph of FIG.

The tubulin polymerization activity regulator according to the present invention is a component obtained by extracting from Ganoderma lingzhi with a solvent containing water (aqueous solvent) or a solvent containing organic solvent (organic solvent). Contains as an active ingredient. Among these, the aqueous solvent extract has an action of inhibiting tubulin polymerization. Further, the organic solvent extract has an action of promoting tubulin polymerization. The “tubulin polymerization activity regulator” includes a tubulin polymerization accelerator and a tubulin polymerization inhibitor.

  Hereinafter, the tubulin polymerization accelerator and its production method, the ganoderma tripenoid compound contained in the tubulin polymerization accelerator and its physical properties, effects and uses will be described. Next, the tubulin polymerization inhibitor will be described in the same manner.

<< Tubulin polymerization accelerator >>
The tubulin polymerization accelerator according to the present invention contains a component extracted from Ganoderma lucidum using a solvent containing an organic solvent (organic solvent).

(Extraction)
Ganoderma lingzhi can be used as a raw material, but the collected natural product (ganoderma) can be processed by drying, crushing, etc. in terms of extraction efficiency. Those prepared in the above are preferred. When the extracted material is pulverized, pulverization by a roll-type pulverizer, a food processor, a ball mill pulverizer, a hammer-type pulverizer, or the like can be exemplified.

  The organic solvent for extraction is preferably a solvent containing 10% or more of an organic solvent as the solvent from the viewpoint of efficiently extracting components such as ganoderma tripenoids having an action of promoting tubulin polymerization. A solvent (organic solvent) containing 50% or more of a solvent is more preferable. In addition, it is desirable that the amount of the organic solvent contained in the organic solvent is usually about 10% or more, more preferably about 30 to 70)% with respect to the total amount of the solvent.

Examples of the organic solvent include alkyl halides (eg, chloroform), alcohols (eg, methanol, ethanol, isopropyl alcohol, etc., C1-10 monovalent aliphatic alcohols, ethylene glycol, glycerin, etc. polyhydric alcohol), hydrocarbons (example: n-hexane, etc., C1-10 approximately aliphatic hydrocarbons), ether (e.g. dimethyl ether, ethyl methyl ether, formula such as diethyl ether, "R ¹ -O-R ² (R ¹ and R ² are alkyl groups of about C1 to 5) ”; aliphatic or aromatic cyclic ethers such as furan, dibenzofuran, and tetrahydrofuran; esters (eg, ethyl acetate, monovalent, etc.) A C1-5 alkyl ester of a polycarboxylic acid) or a mixture of water and these organic solvents. It can gel.

  In particular, methanol, ethanol, and isopropyl alcohol are preferable as the alcohols, but ethanol or hydrous ethanol is particularly preferable from the viewpoint that there is little adverse effect on the human body even if it remains in the extracted components. As the water-containing ethanol, for example, water-containing ethanol having an ethanol concentration of 5 to 99.5% can be used.

  Extraction processing is not particularly limited, but, for example, a natural product collected from Yamano or artificially cultivated ganoderma is dried or pulverized as necessary, and then immersed in an extraction solvent and allowed to stand to extract desired components. Known extraction methods such as an immersion method (cold immersion method, digestion method) and a Soxhlet extraction method by heating reflux using a Soxhlet extractor can be applied. The extract thus extracted may be concentrated or dried by evaporating the solvent, if necessary, and can be prepared in the form of a solid or a powder thereof.

  The extraction temperature is not particularly limited as long as the extraction solvent to be used is not frozen or solidified. Further, the extraction time is not particularly limited as long as a desired component (said tripenoids) can be extracted, and is, for example, 1 hour to 3 days. There is no restriction | limiting in particular also in the pressure of an extraction process, For example, they are normal pressure-4 atmospheres.

  In addition, the extract may be subjected to various types of chromatography such as silica gel column chromatography, reverse phase preparative liquid chromatography, and thin layer chromatography (TLC) to repeat the fractionation operation to increase the content and purity of the extracted components. Good.

(Extracted components)
The following tripenoids (1) to (34) are included as components (reishi organic solvent extract) obtained by extraction from Ganoderma lingzhi with the above organic solvent. The presence of these triterpenoids in the extract can be confirmed by LCMS analysis (liquid chromatograph mass spectrometry) using, for example, LCMS-IT-TOF (Shimadzu Corporation "LCMS-2020").

  The present inventors have found that these tripenoids (1) to (34) (particularly (1) to (6), (9) to (33)) have an action of promoting tubulin polymerization. ing. Therefore, the tubulin polymerization accelerator according to the present invention includes the following tripenoid compounds (1) to (34) (among these, (7), (8) and (34) are excluded. And those containing one or more selected from the group consisting of: These compounds (1) to (34) are commercially available from various manufacturers (Quality Phytochemicals LLC, American Custom Chemicals Corporation, AK scientific, BOC Sciences, Cheminstock, Chromadex, ChemFaces, BioBioPha, etc.) as compounds derived from Reishi. Therefore, it may be purchased and used. Further, for example, (7) Ganoderic acid DM can be obtained commercially from ChromaDex Inc. (Santa Ana, CA).

(1) Ganoderic acid Z (Ganoderic acid Z),

(2) Ganoderic acid N (Ganoderic acid N),

(3) Ganoderic acid S

(4) Ganoderic acid F

(5) Ganoderic acid SZ (Ganoderic acid SZ),

(6) Ganoderic acid LM2

(7) Ganoderic acid DM

(8) Ganoderic acid A

(9) Ganoderic acid AM1

(10) Ganoderic acid B

(11) Ganoderic acid D

(12) Ganoderic acid Y

(13) Ganoderic acid TN

(14) Ganoderic acid TQ

(15) Ganolucidic acid A

(16) Ganodermanontriol

(17) Ganodermanondiol

(18) Ganoderol A

(19) Ganoderol B

(20) Ganoderiol F

(21) Lucialdehyde A

(22) Lucialdehyde B

(23) Ganoderic acid C1

(24) Ganoderic acid C2

(25) Ganoderic acid C6

(26) Ganoderic acid H

(27) Ganoderic acid K

(28) Ganoderic acid TR

(29) Ganoderenic acid A

(30) Ganoderenic acid C

(31) Ganoderenic acid D

(32) Ganoderenic acid F

(33) Ganoderenic acid H

(34) Ganoderic acid DM methyl ester

(Salt, ester)
The extracted compounds (1) to (34) may be esterified by reacting with an alcohol, or may be reacted with a base to form a salt. Examples of esters of these compounds (1) to (34) include linear or branched alkyl esters having 1 to 11 carbon atoms such as methyl esters. Examples of salts of these compounds (1) to (34) include alkali metal salts such as sodium and potassium, ammonium salts and the like.

(Ingredient concentration)
The concentration of the active ingredient contained in the tubulin polymerization accelerator (total of any one of compounds (1) to (6) and (9) to (33)) is bound to tubulin, and the tube It is not particularly limited as long as it exhibits the action of promoting the polymerization of phosphorus, and is adjusted to the dosage form, usage, and dosage in 100% by weight of the tubulin polymerization accelerator so that the blood concentration is 0.1 to 100 μM. And can be appropriately set from the range of 0.1 to 100% by weight.

(Evaluation of tubulin polymerization accelerator)
Conventionally, tubulin polymerization accelerators such as taxol (paclitaxel) exist, but taxol has the following chemical structure (A1) and is a larger molecule than the above compounds (1) to (34), and is taken up into cells. It is thought that it is hard to be repelled. On the other hand, since the compounds (1) to (34) are molecules having a steroid skeleton as a basic skeleton, the molecular size is smaller than that of taxol, and it is considered that they are relatively easily taken up by cells. As a result, the compounds (1) to (34) of Ganoderma tripenoids according to the present invention exhibit stronger tubulin polymerization promoting activity and suppress cell division compared with taxol, and thus prevent mold, anthelmintic, Each effect of weeding, and the effect of treatment of specific diseases (prevention of diseases such as neoplastic diseases, psoriasis, stenosis, myocardial infarction, glomerulonephritis, improvement of symptoms, reduction of transplant rejection), etc. It is speculated that it will be obtained.

  The tubulin polymerization activity (ΔRFU average) is the total including 0 minutes at the start of every 1 minute from the start of the tubulin polymerization reaction using the “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). The average value is shown when the relative fluorescence (RFU) indicating the amount of tubulin polymer is quantified 61 times. Tubulin polymerization activity (ΔRFU60) indicates the RFU value at the 61st determination time point.

Compounds (1) to (34) have the same basic steroid skeleton, but have different chemical structures, so that not only the tubulin polymerization promoting activity is different, but also the cell lethality ( %) Is also different. Therefore, it is desirable that the tubulin polymerization accelerator according to the present invention is comprehensively evaluated based on at least (i) tubulin polymerization activity (IC ₅₀ ) and (ii) cell lethality (%).

  Among these, the cell mortality rate (%) indicates the result that the above compound is actually taken up by the cell to promote or inhibit polymerization of tubulin, or inhibit depolymerization and cause apoptosis in the cell. Yes, it is speculated that “Ease of action on cells” affects.

  In any two compounds (tubulin polymerization accelerator), when the tubulin polymerization accelerator activity is equivalent, the tubulin polymerization accelerator with higher cell lethality (: 100-cell viability%) is more effective for cells. Since it can be said that it acts easily, its usefulness as a tubulin polymerization accelerator is high.

  From this point of view, as shown in Table 1 below, particularly preferred tubulin polymerization accelerators are (14) Ganoderic acid TQ (3) and (3) Ganoderic acid S (Ganoderic acid SQ), respectively. S), (6) Ganoderic acid LM2 (Ganoderic acid LM2), (22) Lucidaldehyde B (12), (12) Ganoderic acid Y, (18) Ganoderol A (Ganoderol A) Yes (see Table 1 below).

(Docking simulation)
The inventors of the present invention describe β-tubulin and 22 types of compounds (1), (3) to (8), (10), (12) to (14), (16) to (24), (28), The docking simulation with (34) was performed by “Molegro Virtual Docker” (manufactured by Northern Science Consulting).

  As a result, the E-total value that is the sum (E1 + E2) of the interaction energy (E1) between each compound and β-tubulin and the internal energy (E2) of the ligand is the minimum in Ganodermic Acid TQ. The value was found (see Table 2). In addition, it means that the smaller the value of E-total (the value is negative (-) and the absolute value is large), the ligand and the protein as the receptor are more stable in the bound state and more likely to interact with each other. Here, the ligand corresponds to each of the 22 types of compounds, and the protein corresponds to β-tubulin, which is an eukaryotic intracellular protein.

  The fact that each 22 types of compounds and β-tubulin are likely to interact means that each of the compounds (1) to (34) containing 22 types of compounds becomes a tubulin of cells (tumor cells, etc.) whose cell proliferation is intense. It means that it can bind and act efficiently and inhibit its growth. This not only means that each of the compounds (1) to (34) is excellent as a tubulin polymerization accelerator, but also elucidates the mechanism that accelerates the polymerization of tubulin and promotes the polymerization of tubulin. This means that it is very significant as a research reagent for screening compounds and their chemical structures.

  As shown in FIG. 5, the E-total value obtained by the docking simulation of each of the 22 types of compounds and β-tubulin has a correlation with the ΔRFU average (see FIG. 5). The E-total value of each compound such as Ganoderic Acid TQ (14) is in the range of −100 to −137, which is higher than the E-total value (-159.3) of paclitaxel. Contains many cells with a higher cell lethality than paclitaxel, and since it has a small molecule, it is more likely to act on cells and is superior to paclitaxel in terms of performance of a tubulin polymerization accelerator (Table 1 above). reference).

Ganodermic Acid TQ, Ganodermic Acid S and Ganoderic Acid TN have chemical structures similar to each other, but have different tubulin polymerization promoting activities.
According to the docking simulation, the oxygen atom of the 15-position acetoxy group of ganoderic acid TQ (Ganoderic acid TQ (14)) is the 276th threonine (Thr 276) and 284th arginine (β-tubulin). Arg284) was found to be hydrogen-bonded (see FIG. 1).

  On the other hand, Ganoderic acid TN (13) having a chemical structure in which the oxygen atom of the carbonyl group of the functional group at position 3 of Ganoderic acid TQ (14) is reduced is Ganoderic acid TN (13). Unlike Q (14), the 15th position acetoxy group and the 370th glycine of β-tubulin (Gly370), the 3rd position hydroxyl group and the 276th threonine of β-tubulin (Thr276), the 26th position It was found that hydrogen bonds were formed between the carboxyl group and the 229th histidine (His229) of β-tubulin (see FIG. 2). However, ganoderic acid TN (13) has a tubulin polymerization promoting activity that is about half that of ganoderic acid TQ (see FIG. 7A), and promotes tubulin polymerization but does not have cytotoxicity.

  Furthermore, for ganoderic acid S (Ganoderic acid SQ) in which the 15-position acetoxy group of the ganoderic acid TQ is changed to hydrogen, the carbonyl group at the 3-position and the 276th position of β-tubulin Threonine (Thr276), a carboxyl group at position 26 and the 27th glutamic acid (Glu27) of β-tubulin were found to have hydrogen bonds (see FIG. 3). However, Ganodermic Acid S has a tubulin polymerization promoting activity that is about half that of Ganodermic Acid TQ (see FIG. 7A), and its cytotoxicity is similar to that of Ganodermic Acid TQ.

  Thus, each of the compounds (1) to (34) including Ganoderic Acid TQ and the like has a similar chemical structure, but each has different tubulin polymerization promoting activity and cytotoxicity. Therefore, it can be said that the compounds (1) to (34) are particularly useful as research tools (research reagents) for elucidating the promotion mechanism of tubulin polymerization.

(Delivery to cells)
An antibody A against at least one of the compounds (1) to (34) is prepared, and an antigen (LAT1, epidermal growth factor receptor or other membrane protein such as CD30 or the like specifically expressed in a target cell (tumor cell or the like) or CD30 And the like (hereinafter referred to as “antibody-drug conjugate”) and the tubulin according to the present invention. It can be considered to be used as a DDS preparation containing a polymerization accelerator (a drug configured to deliver a predetermined drug only to specific cells using a drug delivery system).

  Here, the antibody A specifically functions to hold each of the compounds (1) to (34), and the antibody B specifically binds to a specific protein expressed only in a specific cell (tumor cell or the like). Since the complex of antibodies A and B has both of these functions, each antibody (1) to (34) is bound to the antibody A portion of the complex of antibodies A and B. If a drug complex is used, compounds (1) to (34) can be specifically delivered to specific cells (tumor cells and the like).

  In the case of the above example, between each compound (1) to (34) and the antibody A is a selectively cleavable linker (for example, Seattle Genetics containing a protein sequence that can be selectively cleaved by a specific protease). Linked by a linker, etc.). Since this antibody-drug conjugate is stable in blood, it is selectively delivered to a specific cell (tumor cell or the like) expressing a specific antigen (CD30 antigen or the like) and taken into the cell. Thereafter, each of the compounds (1) to (34) can be released by cleaving the linker with the cell-derived proteolytic enzyme. As a result, each of the compounds (1) to (34) is delivered to a specific cell, and apoptosis can be specifically caused only in the cell (tumor cell or the like).

(Cytotoxicity test)
The cytotoxicity of the tubulin polymerization accelerator can be evaluated by a generally known cytotoxicity test (MTT method). MTT is thiazolyl blue tetrazolium bromide, also known as 3- (4,5-di-methylthiazol-2-yl) -2,5-diphenyltetrazole bromide, yellow tetrazole.

For example, when evaluating the cytotoxicity of the above-mentioned compounds (1) to (34) (100 μM), 100 μL of breast cancer cells (for example, MCF7; 1 × 10 ⁵ cells / mL) are dispensed into each well of a 96-well plate. The 96-well plate into which the breast cancer cells are dispensed is cultured at 37 ° C. for 24 hours in a thermostatic bath containing 5% CO ₂ (solution (A)).

In addition, 1 μL of a sample solution in which the total molar concentration of any one or more of the tripenoid compounds (1) to (34) is 10 mM is added to an EMEM medium (Eagle's minimal essential medium; 99 μL) to prepare a tripenoid-containing medium. The obtained tripenoid-containing medium is added to each well containing the solution (A). Furthermore, the cells are cultured for 72 hours under the same conditions (in a thermostatic bath containing 5% CO ₂ ) (solution (B)).

Next, 20 μL of the MTT solution is added to this solution (B) to the solution (B) in each well and allowed to stand for 4 hours under the same conditions (solution (C)).
Next, the solution (C) was discarded, and then 100 μL of isopropanol hydrochloride (anhydrous isopropanol added with 0.04 to 0.1 N HCl) was added, so that an insoluble blue formazan dye converted by living cells was obtained. The cell viability can be calculated by examining the wavelength (absorbance) at 570 nm using a device (Micro Reader, manufactured by BioTech, model number: ELx800TM), and quantifying this. The rate (: 100-% cell viability) can be calculated.

As described above, the evaluation of the cytotoxicity of the tubulin polymerization accelerator is an index representing the ease of action of the tubulin polymerization accelerator on the cells.
Although MTT is a yellow substance, it is reduced by intracellular mitochondrial reductase to produce insoluble blue formazan. Cell viability (= 100−cell lethality%) is determined by utilizing the fact that viable cells develop a blue color. Usually, dimethyl sulfoxide, acidic ethanol solution, or dilute hydrochloric acid solution of sodium dodecyl sulfate (SDS) is added to solubilize the insoluble formazan dye. Quantification is performed by measuring the absorbance of the obtained colored solution at an arbitrary wavelength (usually between 500 and 600 nm) with a spectrophotometer. The absorption maximum wavelength depends on the solvent used.

(Measurement of tubulin polymerization promoting activity)
The tubulin polymerization promoting activity can be expressed by how much ΔRFU of each compound is higher than the ΔRFU (ΔRFU average or ΔRFU60) of the control substance not containing a substance that affects tubulin polymerization. It can also be evaluated as the amount of tubulin polymerization promoter (ie, IC ₅₀ ) that inhibits 50% of the resulting cell growth in the absence of other agents that affect phosphopolymerization activity. For example, in the cytotoxicity test described above, a plurality of samples having different final concentrations are prepared for the same tubulin polymerization accelerator (for example, 100 μM, 75 μM, 50 μM, 25 μM, and 12.5 μM samples are prepared), and the cytotoxicity is measured. A 50% cell growth inhibitory concentration (IC ₅₀ ) can be calculated by obtaining an approximate curve from the relationship between the concentration of these samples and cytotoxicity.

  Tubulin polymerization activity (ΔRFU average, ΔRFU60) can be measured by using a tubulin polymerization assay kit (Cytoskeleton Inc., Denver, CO, USA). ΔRFU average means the average of RFU values per unit time (1 minute) measured with the kit, and ΔRFU60 shows the RFU value at 60 minutes after the start of polymerization of tubulin when measured with the kit. .

  Specifically, the tubulin polymerization activity was measured by placing a tubulin polymerization accelerator and a blank solution (blank only in a solvent) at 37 ° C., respectively, and 50 μL of a reaction mixture (1 × Buffer 1 (80 mM Piperazine-N, N'-bis [2-ethanesulfonic acid] sequisodium salt; 2.0 mM Magnesium chloride; 0.5 mM Ethylene glycol-bis (b-amino-ethyl ether) N, N, N ', N' -tetra-acetic acid, pH 6.9, 10 μM fluorescent reporter.), 10% Glycerol, 1 mM GTP, 2 mg / mL Tublin), and plot the fluorescence intensity (value at 420 nm) of the changing reaction solution over time Is the method. The fluorescence (420 nm) indicating the amount of the tubulin polymer at this time is expressed as a relative fluorescence unit (RFU), and the ΔRFU average and ΔRFU60 can be calculated from the RFU.

  By examining the change over time in the amount (RFU) of this tubulin polymer, it is possible to ascertain the time until the tubulin depolymerization inhibitor has a sufficient inhibitory effect on tubulin depolymerization (tubulin polymerization acceleration effect). It is possible to know how much immediate action (or slow action) it has compared to other drugs (paclitaxel or the like).

<< Use of tubulin polymerization accelerator >>
The tubulin polymerization accelerator according to the present invention can be used as food (supplements, food additives, etc.), cosmetics, pharmaceuticals, research reagents and the like. These foods and the like contain one or more of the above tripenoid compounds (1) to (6) and (9) to (33). Furthermore, foods and the like may optionally contain compounds (7), (8), and (34).

[Food]
As described above, the food according to the present invention contains the compounds (1) to (6) and (9) to (33) as tubulin polymerization accelerators, which are contained in a high concentration in the ganoderma organic solvent extract. Any one or two or more compounds are contained, and optionally compounds (7), (8) and (34) are contained.

  For example, since ganoderol B (19) has 5α-reductase inhibitory activity and male hormone receptor binding activity, it is expected to be effective for prostatic hypertrophy. Ganoderol B is effective for diabetes because it has α-glucosidase inhibitory activity. Therefore, if it is a food containing Ganoderol B, in addition to the effects of prevention, reduction or treatment of the above-mentioned diseases (tumor diseases etc.) due to the tubulin polymerization promoting effect, prostatic hypertrophy and diabetes are prevented, reduced or reduced. It is useful as a food that can be treated.

  Moreover, ganoderic acid DM (7), ganoderic acid F (4), and gadermanone diol (17) show the osteoclast differentiation inhibitory effect and can be expected to be effective for osteoporosis. Therefore, if it is a food containing these compounds (7), (4) and (17), in addition to the effect of preventing, reducing or treating the aforementioned diseases (tumor diseases, etc.) due to the tubulin polymerization promoting effect. Therefore, it can be said that it is useful as a food product that has the effect of preventing, reducing or treating osteoporosis.

  Ganoderic acid A (29) has aldose reductase inhibitory activity and is effective for diabetic complications. Therefore, if it is a food containing these compounds (29) as an active ingredient, in addition to the effects of prevention, reduction or treatment of the aforementioned diseases (tumor diseases etc.) due to the tubulin polymerization promoting effect, diabetes is prevented, It can be said that it is useful as a food product that provides an effect of reducing or treating.

  As a dosage form of food according to the present invention, any one or more of Ganoderma organic solvent extract or compounds (1) to (6) and (9) to (33) are used as food as they are. What was prepared can be mentioned. As a form of the food according to the present invention, any one or more of Ganoderma organic solvent extract or compounds (1) to (6) and (9) to (33) are used as another food. Arbitrary forms usually used, such as those added or those in the form of food or health food such as capsules and tablets, can be exemplified.

  In addition, when the tubulin polymerization accelerator according to the present invention is blended in foods and ingested by humans or other animals, excipients, extenders, binders, thickeners, emulsifiers, colorants are added to the foods. A fragrance, a food additive, a seasoning and the like can be appropriately added together with a tubulin polymerization accelerator. In that case, the tubulin polymerization accelerator according to the present invention can be mixed with food and these optional components, and the dosage form can be formed into a powder, granule, tablet or the like depending on the application. Furthermore, a tubulin polymerization accelerator can be mixed in a food raw material to prepare a food, which can be commercialized as a functional food.

  In a blood concentration in a human or the like who ingested the food, the total concentration of any one or more of the compounds (1) to (6) and (9) to (33) is 0.01 to 100 μM. It is preferable that the above compounds (1) to (6) and (9) to (33) are contained in food. For example, an example of ingesting one or more of compounds (1) to (6) and (9) to (33) or the above extract in an aspect of 1 mg to 1000 mg / day for an adult (body weight 60 kg). Can be mentioned.

(supplement)
The tubulin polymerization accelerator according to the present invention can be used as a supplement. When using as a supplement, for example, any one or more of compounds (1) to (6) and (9) to (33), or a reishi organic solvent extract is used as a raw material, and a known excipient is used. It can be used in the form of granules, powders, gels, capsules, pastes or tablets by conventional methods. Among these, when making into a paste, a tubulin polymerization accelerator may be included in the pores of cyclodextrin. Moreover, you may make the said raw material contain a compound (7), (8) or (34) arbitrarily.

  The total content of any one or more of Ganoderma tripenoid compounds (1) to (6) and (9) to (33) in the supplement is not particularly limited, but about 200 mg of Ganoderin in 100 g of Ganoderma Since an acid is contained and 25 to 30 g of ganoderma is usually used in a single intake, for example, in one supplement (1 g), for example, compounds (1) to (6) and (9) to It is preferable that at least one of (33) is contained in a total amount of about 5 mg to 60 mg.

(Food additive)
Compounds (1) to (6) and (9) to (33) exhibit a strong bitter taste and suppress cell growth, so that bitterness is permitted as a food additive (bitterings and fungicides). It can be used for soft drinks and alcoholic beverages. Compounds (1) to (6) and (9) to (33) can be handled basically in the same manner as known bittering agents such as sesquiterpenes.

  Conversely, it can also be used as an antifungal agent that suppresses the bitterness of the compounds (1) to (6) and (9) to (33). In that case, a bitterness masking agent (Kao "Benecoat BMI-40" The above effects (cancer prevention effect based on tubulin polymerization activity promotion, prevention of diseases such as psoriasis, stenosis, myocardial infarction, glomerulonephritis, improvement of symptoms, reduction of transplant rejection, etc. ) And may be used as a food additive having such a function.

  When using the compounds (1) to (6) and (9) to (33) as the food additive, the following other food additives permitted in terms of safety are used in combination with water-soluble or oil-soluble compounds. May be used. Other food additives include monohydric alcohols (such as ethanol), polyhydric alcohols (eg, ethylene glycol, glycerin, etc.), animal and vegetable oils (eg, glycerin mono, di, tri-fatty acid esters). A known solvent such as a fatty acid ester of a monohydric alcohol), a known surfactant or a known emulsifier {eg sugar ester, sorbitan fatty acid ester (sorbitan ester), propylene glycol fatty acid ester (PG ester), lecithin)}, etc. Can be mentioned.

  Further, the compounds (1) to (6) and (9) to (33) are emulsified together with water / natural gums and a high molecular weight substance such as polysaccharides using a conventional solvent or emulsifier to obtain an oily food (mayonnaise, etc.) ), Water-based foods (soft drinks, etc.).

  The total content of any one or more of compounds (1) to (6) and (9) to (33) in the food additive is not particularly limited, but about 200 mg of ganoderic acid in 100 g of ganoderma , Lashishi itself is sufficiently bitter, and 25-30 g of Ganoderma is usually used in a single intake (50 mg to 60 mg in terms of ganoderic acid). In the package as a single dose, any one or more of compounds (1) to (6) and (9) to (33) are preferably contained in a total amount of 10 mg to 60 mg.

[Cosmetics]
The cosmetic according to the present invention contains at least one of Ganoderma tripenoid compounds (1) to (6) and (9) to (33). This cosmetic may optionally contain compound (7), (8) or (34). Since Ganoderma tripenoid compounds (1) to (6) and (9) to (33) have a basic skeleton of steroids, cellular lipids and compounds (1) to (6) and (9) to (33) It is preferable to be used as a (skin) cosmetic because it is easily taken into cells from the skin due to interaction between lipids. This cosmetic is useful for suppressing and preventing skin tumors (such as warts) and hair cell growth, and has uses for antitumor agents and hair suppressants.

  The final concentration of the above-mentioned Ganoderma tripenoid compound or Ganoderma's solvent (or hot water (hot water)) extract in the cosmetic according to the present invention is not particularly limited, but for example, the effective ingredient is from 0.1 μM, for example. It is preferable to include in cosmetics so that it may become 100 micromol, More preferably, it is 15-25 micromol. In this case, in order to efficiently penetrate the extracted components applied to the skin into the cells, penetration enhancers (transdermal absorption) such as dimethyl sulfoxide (DMSO), ion activator, ethanol, propylene glycol, fatty acid, fatty acid ester, etc. Accelerators) may be included as cosmetic ingredients. Among these, when using DMSO, it is preferable to contain 10 weight% or less with respect to the whole cosmetics. When the above-described extraction is performed using ethanol, ethanol is included again as a cosmetic component, which is advantageous in that it is not necessary to remove the ethanol used for extraction.

  Examples of cosmetic dosage forms include solution systems, solubilization systems, emulsification systems, powder dispersion systems, water-oil two-layer systems, water-oil-powder three-layer systems, gels, mists, sprays, mousses, roll-ons, sticks, etc. In addition, a sheet impregnated or coated on a sheet such as a nonwoven fabric can be used.

  In the present invention, any one or more of the compounds (1) to (6) and (9) to (33) includes oils and fats such as vegetable oil, waxes such as lanolin and beeswax, hydrocarbons, fatty acids, and higher alcohols. , Esters, various surfactants, pigments, fragrances, vitamins, plant and animal extract components, UV absorbers, antioxidants, preservatives, bactericides, etc. Good.

[Anti-mold agent (for grass)]
The antifungal agent (for grass) according to the present invention contains at least one of the above compounds (1) to (6) and (9) to (33) as an active ingredient of a fungicide, As with fungicides (eg, isothiazoline fungicides), a small amount is sprayed on the leaves of ornamental plants, crops and other young seedlings to prevent the growth of microorganisms attached to the plant surface. Here, the said fungicide (for grass) may contain the said compounds (7), (8), (34). In general, when a person eats a crop sprayed with a fungicide, the adverse effect on the human body of the active ingredient of the fungicide becomes a problem, but as described above, the active ingredient of the fungicide according to the present invention Since the above-mentioned Ganoderma-derived tripenoid compounds (1) to (6), (9) to (33) and organic solvent extracts from Ganoderma are safe ingredients that can be used as food. There is no negative effect on the human body. The fungicide (for grasses) according to the present invention preferably contains the above-mentioned active ingredient in a final concentration of, for example, 1 μM to 100 μM. This fungicide has a solvent such as DMSO as described above. It may be included.

[Research reagents]
Since Ganoderma tripenoid compounds (1) to (34) have different tubulin polymerization promoting activities due to differences in their chemical structures, each compound (1) to (34) has a mode of binding to β-tubulin and a tube. It is thought that the expression mechanism of phosphorylation-promoting activity (effect on signal transduction system, etc.) is different, but the details are currently unknown. Therefore, the compounds (1) to (34) having different degrees of tubulin polymerization promoting activity can be used as research reagents for elucidating the tubulin polymerization promoting mechanism and its influence on signal transmission.

  Moreover, since each compound (1)-(34) has a different chemical structure as described later, the binding mode with tubulin and the tubulin polymerization promoting activity are different. Therefore, based on these compounds (1) to (34), it is possible to develop a more active tubulin polymerization promoting activator, and it can also be used as a research reagent for this purpose.

  As an example of the former, there can be mentioned an example in which gene expression and signal transduction are comprehensively analyzed with a DNA microarray or the like when each of the compounds (1) to (34) is used.

  Specifically, among each of the compounds (1) to (34), ganodermic acid TQ (compound (14)) having the strongest tubulin polymerization promoting activity and the weakest ganodermic acid N (compound ( 2)) is cultured in a medium containing the same molar concentration (0.01 μM to 100 μM, for example, 30 μM), and each target cell is cultured, and mRNA is extracted from the cultured cells. An example of comprehensive analysis of cell gene expression can be given. Further, as the latter example, the degree of tubulin polymerization promoting activity and the binding mode with β-tubulin are different among the compounds (1) to (34) and also different from taxol. It can be used as a reference reagent having tubulin polymerization promoting activity. This also contributes to the discovery of antitumor active anticancer agents.

<< Tubulin polymerization inhibitor >>
Medicinal mushrooms such as Ganoderma lingzhi have long been believed to have a folklore-like function to enhance biological defense responses (immune function), and people with compromised immune functions such as tumors, allergic patients, and the elderly Has been considered useful. Ganoderma contains β-glucan as a useful component. β-glucan is a high-molecular polysaccharide formed by connecting glucose (glucose) contained in mushrooms and the like, and the binding mode of glucose is slightly different depending on the type of mushroom.

The present inventors have found that a hot water extract of Ganoderma lingzhi has a tubulin polymerization inhibitory activity, and completed a tubulin polymerization inhibitor. Therefore, the tubulin polymerization inhibitor according to the present invention contains, as an active ingredient, a component obtained by extracting water from a ganoderma lingzhi with a solvent containing water (aqueous solvent), particularly β-glucan. It has the effect | action which inhibits polymerization activity.

(Aqueous solvent extraction)
Reishi ( Ganoderma lingzhi ) as a material used for aqueous solvent extraction may be subjected to extraction processing of natural products and artificial cultures collected in Yamano as it is. Preferable examples include those prepared by processing such as (freezing) drying and crushing.

  The extraction solvent may be a solvent containing water (aqueous solvent), and an extraction solvent that can inhibit the polymerization of tubulin can be obtained as long as the extraction solvent contains 50 to 100% of water. To preferred. Examples of such a solvent include water, a buffer solution (for example, PBS, Tris-HCl), a 30% to 100% ethanol aqueous solution, and the like.

  Typically, the active ingredient is obtained by heating and extracting Ganoderma under the following predetermined extraction conditions in the presence of an aqueous solvent, filtering the resulting extraction mixture, and distilling off the solvent from the filtrate. To obtain an extract of The extraction conditions can be changed as appropriate.

  The extraction temperature is not particularly limited as long as a component mainly containing polysaccharides (β-glucan, peptidoglycan, arabinoxyl glycan, and other mannitol) can be extracted from ganoderma, and is 30 ° C to 125 ° C, preferably 80 ° C to 125 ° C. C, most preferably around 121 ° C. (115 ° C. to 125 ° C.) can be used.

  The pressure at the time of extraction is not particularly limited as long as a component mainly containing polysaccharides (β-glucan, etc.) can be extracted from ganoderma, and may be normal pressure, but when pressurized, for example, 1060 hPa to 4900 hPa (about 1 to 5 atm) And particularly preferably 2 atm.

The extraction time is not particularly limited as long as a component mainly containing a polysaccharide (β-glucan or the like) can be extracted, and is, for example, several minutes to 3 days, and most preferably about 20 minutes.
Furthermore, a lower alcohol such as ethanol or acetone is added to the above extract, the precipitate is neutralized and subjected to membrane filtration, and proteins other than polysaccharides mainly containing β-glucan are precipitated and fractionated. Good. The above extract may be further subjected to ultrafiltration treatment, ultrasonic treatment, etc., and such ultrafiltrate (filtrate and filtrate), ultrasonic treatment product, etc. It is an extract and is included in the present invention.

  In addition, the above extract is further subjected to fractionation (separation / purification) by various types of chromatography such as silica gel column chromatography, reverse phase preparative liquid chromatography, thin layer chromatography (TLC), and high performance liquid chromatography. The content and purity of the active ingredient in the extract may be increased by freeze-drying or the like.

(Measurement of tubulin polymerization inhibitory activity)
Tubulin polymerization inhibitory activity should be assessed as the amount of tubulin polymerization inhibitor that inhibits 50% of cell growth that occurs in the absence of other drugs that affect tubulin polymerization activity (ie, IC ₅₀ ). Can do. For example, in the cytotoxicity test described above, a plurality of samples having different final concentrations are prepared for the same tubulin polymerization inhibitor (for example, 100 μM, 75 μM, 50 μM, 25 μM, and 12.5 μM samples are prepared), and the cytotoxicity is measured. A 50% cell growth inhibitory concentration (IC ₅₀ ) can be calculated by obtaining an approximate curve from the relationship between the concentration of these samples and cytotoxicity. A lower IC ₅₀ value indicates higher activity as a polymerization inhibitor (higher cytotoxicity).

  Moreover, the measurement of the tubulin polymerization inhibitory activity over time can be examined using, for example, a tubulin polymerization assay kit (Cytoskeleton Inc., Denver, CO, USA). A tubulin polymerization inhibitor and blank solution (blank is solvent only) were each incubated at 37 ° C, and 50 µL of reaction mixture (1 x Buffer 1 ((80 mM Piperazine-N, N'-bis [2-ethanesulfonic acid] sequisodium salt; 2.0 mM Magnesium chloride; 0.5 mM Ethylene glycol-bis (b-amino-ethyl ether) N, N, N ', N'-tetra-acetic acid, pH 6.9, 10 μM fluorescent reporter ), 10% Glycerol, 1 mM GTP, 2 mg / mL Tublin) and plot the fluorescence (420 nm) of the changing reaction solution over time, where the fluorescence (420 nm) is the relative fluorescence unit. (RFU).

<< Use of tubulin polymerization inhibitor >>
The tubulin polymerization inhibitor according to the present invention can be used as food (supplements, food additives, etc.), cosmetics and the like. These contain an extract (reishi water-based solvent extract) obtained from the above-described extraction from reishi with an aqueous solvent.

[Food]
The food according to the present invention contains the above-described reishi water-based solvent extract, and can be expected to have each effect of preventing or reducing diseases related to cell proliferation. Examples of the food according to the present invention include those prepared as such as the above-mentioned Ganoderma hydrate solvent extract, those added to other foods, or any form commonly used for food or health food such as capsules and tablets. be able to.

  In addition, when ingesting or administering the above-described reishi water-based solvent extract in food, the reishi water-based solvent extract is appropriately mixed with an excipient, a bulking agent, a binder, a thickener, an emulsifier, It can be mixed with a coloring agent, a fragrance, a food additive, a seasoning, etc., and formed into a powder, granule, tablet or the like according to the use. Furthermore, it can be ingested by mixing the above-mentioned reishi water-based solvent extract into a food material to prepare a food and commercializing it as a functional food. The food can be produced by adding the ganoderma-based solvent extract to the food in an amount such that the concentration of the ganoderma-based solvent extract is 0.1 to 100 μM in blood concentration in a human or the like who has ingested the food. preferable.

(supplement)
The tubulin polymerization inhibitor according to the present invention can be used as a supplement. In the case of a supplement, for example, the above-mentioned Ganoderma hydrate solvent extract is made into a granule, powder, gel, capsule, paste or tablet by a conventional method using a known excipient as a supplement. Can be used. Among these, in the case of making a paste, a tubulin polymerization accelerator may be included in the cyclodextrin. Although there is no restriction | limiting in particular in content of the said extract in a supplement, For example, it is desirable to make it contain in a supplement with the component quantity that a blood concentration will be 8-12 microgram / mL.

  It is desired that 100 g of Ganoderma contains about 55 g of β-glucan, and it is desired to ingest 25 g to 30 g of Ganoderma in a single ingestion. It is desirable to include a total of 14 to 20 g. For example, 4 supplements containing 5 g of the above extract may be taken as one intake.

(Food additive)
In addition, the above reishi water-based solvent extract has been used as a traditional Chinese medicine since ancient times, and is excellent in safety and suppresses cell growth. Therefore, as a food additive (an antifungal agent), soft drinks, alcoholic beverages, etc. It can be added to and used. When using as a food additive, the above extract may be made oil-soluble using a known solvent such as alcohol, polyhydric alcohol, animal or vegetable oil, a known surfactant or a known emulsifier, It may be used after emulsification with a common solvent or emulsifier together with water, natural gums and polysaccharides. You may include the said extract made oil-soluble by this process in oil-based foodstuffs (mayonnaise etc.) or an aqueous foodstuff (soft drink etc.) as it is.

[Cosmetics]
The cosmetic according to the present invention contains the above reishi water-based solvent extract. The above-mentioned reishi water-based solvent extract is preferably used as a cosmetic because it inhibits tubulin polymerization. This cosmetic can be expected to be useful for suppressing and preventing skin tumors (such as warts) and fungal growth in the skin. The cosmetic according to the present invention preferably contains the above components in the cosmetic so that the final concentration is, for example, 0.1 μM to 100 μM. In this case, in order to efficiently penetrate the extracted components applied to the skin into the cells, penetration enhancers (transdermal absorption) such as dimethyl sulfoxide (DMSO), ion activator, ethanol, propylene glycol, fatty acid, fatty acid ester, etc. Accelerators) may be included as cosmetic ingredients.

  In addition to the above components, the cosmetics according to the present invention include oils and fats such as vegetable oils, waxes such as lanolin and beeswax, hydrocarbons, higher alcohols, various surfactants, pigments, fragrances, vitamins, plants, Animal extract components, ultraviolet absorbers, antioxidants, fungicides / preservatives (excluding Ganoderma solute extract), bactericides, and the like may be appropriately blended.

  The dosage form of the cosmetic is not particularly limited, and examples thereof include a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, a gel, a mist, and a spray. In addition to mousse, roll-on, sticks, etc., a sheet such as a nonwoven fabric impregnated or coated may be used.

[Anti-mold agent (for grass)]
The antifungal agent (for grasses) according to the present invention contains the above-mentioned reishi water-based solvent extract as an active ingredient of the antifungal agent, and unlike general antifungal agents, for example, seedlings such as ornamental plants and crops A small amount is sprayed on the leaf surface to prevent the growth of fungi and other microorganisms adhering to the surface. When sprayed on crop seedlings, when humans eat crops that use herbicides, the negative effects on the human body of the active ingredients of the fungicide will be a problem. There is no negative effect on the human body. It is desirable that the fungicide according to the present invention (for grasses) contains, as a final concentration, an extract extracted with the above aqueous solvent at, for example, 1 μM to 100 μM.

Since this fungicide has a high water content, it suppresses the growth of phytopathogenic fungi ( Aspergillus family) and other phytopathogenic fungi that parasitize plants and cause illness. It is expected that the action will work and an antifungal effect will be obtained.

  In the above description, either tubulin polymerization inhibition action (in the case of aqueous solvent extraction) or promotion action (in the case of organic solvent extraction) using either an aqueous solvent or an organic solvent from Ganoderma However, in the present invention, an arbitrary amount ratio of the aqueous solvent and the organic solvent insoluble in the aqueous solvent is combined. Solvent extraction is performed using a mixed solvent (eg, water / chloroform 50% each), and components dissolved in each layer (eg, water layer, chloroform layer) are separated and used with a separatory funnel or the like. Good.

[Test Example 1]
<< Cytotoxicity test of Ganoderma triterpenoids (compounds (1) to (34)) >>
In Test Example 1, the cytotoxicity of 34 types of triterpenoids (compounds (1) to (34)) derived from Ganoderma lingzhi having different chemical structures on human breast cancer cells (MCF7) was examined.

(experimental method)
The cytotoxicity of Paclitaxel, Vinblastine, and Ganoderma lingzhi- derived triterpenoids (compounds (1) to (34)) to breast cancer cells (MCF7) was examined by the MTT method.

That is, the MCF7 cell solution (EMEM medium) was seeded at 1.0 × 10 ⁴ cells / well in each well of the EMEM medium composed of a 96-well plate. After culturing under conditions of 5% CO ₂ and 37 ° C. for 24 hours, the compound (1) to (34), paclitaxel, or vinblastine was added as a sample together with the medium exchange. For the control, no compound such as a sample was added, and only the cultured liquid was used.

Thereafter, after culturing at 5% CO ₂ · 37 ° C. for 72 hours, 20 μL of MTT solution was added to each well and cultured at 5% CO ₂ · 37 ° C. for 4 hours. The medium was discarded, 100 μL of isopropanol hydrochloride solution was added to each well, and the mixture was allowed to stand at room temperature for 4 hours under light-shielding conditions, and then the absorbance at 570 nm was measured. Cytotoxicity was measured for each sample at five final well concentrations of 100 μM, 75 μM, 50 μM, 25 μM, and 12.5 μM, and an approximate curve was obtained to calculate a 50% cell growth inhibitory concentration (IC ₅₀ ). The results are shown in Table 3 below. This IC ₅₀ represents tubulin polymerization promoting activity, and a smaller value indicates higher activity.

(Results and discussion)
Table 3 below shows the chemical structures of the compounds (1) to (34) subjected to the experiment, the names of the compounds, and the cytotoxicity results obtained. Compounds with a cell growth rate of 50% compared to the control group having a concentration of 100 μM or less are Ganodermic Acid TQ (14), Ganodermic Acid S (3), Lucidaldehyde B (22), Gano There were six types, derrick acid LM2 (6), ganoderic acid Y (12), and ganoderol A (18). Ganodermic acid TQ (14) showed strong cytotoxicity, but ganodermic acid TN (13) was not cytotoxic. Moreover, although ganoderol A (18) showed strong cytotoxicity, ganoderol B (19) was not confirmed to be cytotoxic. Furthermore, the 3-position of many other ganoderma triterpenoids that showed cytotoxicity was a carbonyl group. From these facts, it is considered that the 3rd position of Ganoderma triterpenoid is a carbonyl group in order to show cytotoxicity. In addition, since most of the compounds that did not show cytotoxicity have a carbonyl group at the 11-position, this substituent may be involved in the presence or absence of cytotoxicity.

  From the above, in relation to the objects and effects of the present invention, the above six kinds of compounds are very excellent as tubulin polymerization accelerators. In particular, the compounds (3) to (7), (10), (12), (14), (16) to (23), (25), which have significant differences in cytotoxicity, including the above six types of compounds About (28), (30)-(32), it acts on a cell equivalent to or higher than paclitaxel to inhibit depolymerization of tubulin (accelerate polymerization), leading to apoptosis and killing the cell. Therefore, it can be said that it is useful as a tubulin polymerization inhibitor having an effect equivalent to or higher than that of paclitaxel.

  Conversely, the compounds (1), (2), (8), (9), (11), (13), (15), (24), (26), (27) that did not show the above significant difference. , (29) and (33) are compounds that do not exhibit cytotoxicity, but are compounds that inhibit tubulin polymerization, and are particularly useful as research reagents for investigating the mechanism of action resulting in such results. Useful.

(Each compound name: Ganoderic acid Z (1), Ganoderic acid N (2), Ganoderic acid S (3), Ganoderic acid F (4), Ganoderic acid SZ (5), Ganoderic acid LM2 (6), Ganoderic acid DM (7), Ganoderic acid A (8), Ganoderic acid AM1 (9), Ganoderic acid B (10), Ganoderic acid D (11), Ganoderic acid Y (12), Ganoderic acid TN (13), Ganoderic acid TQ ( 14), Ganolucidic acid A (15), Ganodermanontriol (16), Ganodermanondiol (17), Ganoderol A (18), Ganoderol B (19), Ganoderiol F (20), Lucialdehyde A (21), Lucialdehyde B (22), Ganoderic acid C1 (23), Ganoderic acid C2 (24), Ganoderic acid C6 (25), Ganoderic acid H (26), Ganoderic acid K (27), Ganoderic acid TR (28), Ganoderenic acid A (29), Ganoderenic acid C (30), Ganoderenic acid D (31), Ganoderenic acid F (32), Ganoderenic acid H (33), Ganoderic acid DM methyl ester (34))

[Test Example 2]
(Evaluation of tubulin polymerization promoting activity of Ganoderma triterpenoids)
Focusing on the influence of 34 types of triterpenoids (1) to (34) derived from Ganoderma lingzhi on the degree of tubulin polymerization, a comprehensive search for tubulin depolymerization inhibiting substances and their structure-activity relationship Accumulated knowledge. In the experiment, 34 types of triterpenoids (compounds (1) to (34)) derived from Ganoderma shown in Table 3 were used, and paclitaxel (Taxol) was used as a positive control.

(experimental method)
The tubulin depolymerization inhibitory activity (tubulin polymerization promotion activity) was evaluated using the “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.) for triterpenoids derived from each ganoderma. After adding buffer and tubulin to a 96-well plate, the final concentration in the system was 2.73 μM for paclitaxel (positive control), and 18.2 μM for each ganoderma triterpenoid (compounds (1) to (34)). It added to the reaction system so that it might become. Each Ganoderma triterpenoid was dissolved in a mixed solution of pure water: DMSO = 9: 1. The buffer contains fluorescent substance DAPI (excitation light wavelength (Ex) = 360 nm, fluorescence wavelength (Em) = the excitation intensity at 360 nm and the fluorescence intensity at 420 nm increase by binding to microtubules, which are tubulin polymers. 420 nm), and in Test Example 2, the degree of tubulin polymerization in each well was measured by measuring the fluorescence intensity. The fluorescence intensity was measured every minute from 0 to 60 minutes after the start of measurement, and was measured 61 times in total.

(Results and discussion)
Table 4 below shows the average value (ΔRFU average) in 61 measurements of the obtained fluorescence intensity RFU for each compound. Moreover, the time-dependent change of the fluorescence intensity RFU in the tubulin polymerization reaction including each compound (1)-(16) is shown in FIG.7 and FIG.8. All 34 types of Ganoderma triterpenoids (compounds (1) to (34)) showed higher fluorescence intensity than the negative control, that is, tubulin polymerization accelerating activity, and the most active compound was Ganodermic Acid TQ ( 14). Ganodermic acid TQ (14) is a compound in which the carbon at position 15 of ganodermic acid S (3) is acetoxylated (see FIG. 1 and Table 3). Since there is a large difference, it is considered that the tubulin polymerization promoting activity of ganoderic acid TQ is strongly related to the acetoxy group at position 15 as an active site. Also, as a whole, a compound having a double bond between the 7th and 8th carbons and a 9th and 11th carbon has strong activity, and a compound having a double bond between the 8th and 9th carbons is active. A weak tendency was seen.

Therefore, it was possible to sufficiently confirm that the compounds (1) to (16) had tubulin polymerization promoting activity and could be used as a tubulin polymerization accelerator.
In addition, as shown in FIGS. 8 and 9, the amount of tubulin polymer (RFU) increased from 12 to 18 minutes after the start of polymerization of tubulin (0 minutes) for compounds (1) to (16). In contrast to paclitaxel, the amount of tubulin polymer (RFU) starts to increase from the point when 4 to 5 minutes have passed. From these facts, compounds (1) to (16) may promote tubulin polymerization by a mechanism different from that of paclitaxel.

  Therefore, in relation to the purpose and effect of the present invention, it can be said that it is useful as a research reagent for inhibiting tubulin polymerization and elucidating the mechanism.

[Test Example 3]
(Docking simulation of Ganoderma derived triterpenoids and tubulin)
It is known that tubulin polymerization accelerators such as paclitaxel (taxol) exhibit their activity by interacting with tubulin. As for triterpenoids derived from Ganoderma lingzhi (compounds (1) to (34)), the tubulin polymerization promoting activity is similar to that in the interaction with paclitaxel (taxol) as in the case of interaction with paclitaxel (taxol). It is possible that the protein is expressed by interacting with a site similar to “the binding site”).

  In the present invention, among the 34 types of triterpenoids (compounds (1) to (34)) derived from Ganoderma lucidum shown in Table 3, 22 types of compounds (Ganoderic Acid Z (1)) whose steric structures are completely identified , Ganodermic Acid S (3), Ganoderic Acid F (4), Ganoderic Acid SZ (5), Ganoderic Acid LM2 (6), Ganodermic Acid DM (7), Ganodermic Acid A (8), Ganode Derrick Acid B (10), Ganodermic Acid Y (12), Ganodermic Acid TN (13), Ganodermic Acid TQ (14), Ganodermanon Triol (16), Ganodermanone Diol (17), Ganoderol A (18), Ganoderol B (19), Ganodeliol F (20), Lucidaldehyde A (21), Lucial Hyd B (22), Ganodermic Acid C1 (23), Ganoderic Acid C2 (24), Ganoderic Acid TR (28), Ganoderic acid DM methyl ester (34)), software “Molgro Virtual Docker” (Northern Science Consulting Inc.), a docking simulation of β-tubulin, which is known to bind to paclitaxel (taxol), was performed.

(experimental method)
Docking simulation was performed using the crystal structure of paclitaxel (taxol) and tubulin (PDB code 1JFF). Originally, paclitaxel binds to β-tubulin, which is a protein, as a binding substance (ligand). In Test Example 3, tripenoids (1), (3) to (8), (10) are used as ligands. , (12) to (14), (16) to (24), (28) and (34) are each docked to β-tubulin, and the energy of interaction of each ligand with the protein during this simulation By calculating the E-total value that is the sum (E1 + E2) of (E1) and the internal energy (E2) of each ligand, each ganoderma triterpenoid (compounds (1), (3) to (8) , (10), (12) to (14), (16) to (24), (28), (34)) and the strength of the interaction between tubulin and the like.
In addition, it represents that a ligand and protein are easy to interact, so that the value of E-total is small (: The sign is negative (-) and the absolute value is large). Table 5 shows the E-total value of each compound.

(Results and discussion)
Among all ganoderma triterpenoids (1), (3) to (8), (10), (12) to (14), (16) to (24), (28), (34), The E-total value of derrick acid TQ (14) was minimized (see Table 5).

From the binding mode of paclitaxel (taxol) and tubulin (above chemical formula, FIG. 4), it can be seen that paclitaxel (taxol) has a hydrogen bond with threonine at the 276th amino acid residue of β-tubulin.

  On the other hand, regarding the mode of binding of ganoderic acid TQ (14) and tubulin (the above chemical formula [A3], FIG. 1), ganoderic acid TQ (14) is mediated by its 15-position acetoxy group. Similarly, since it has a hydrogen bond with the threonine of the 276th amino acid residue of β-tubulin, the acetoxy group at the 15th position of ganoderic acid TQ (14) serves as an active site for promoting tubulin polymerization. The results were confirmed to be involved.

  In addition, regarding the binding mode of ganoderic acid TN (13), which is also a compound having an acetoxy group at the 15-position (Fig. 2), The only difference is that the 3rd position is the Carbonyl group in the former, and the 3rd position is the Hydroxy group in the latter. Nevertheless, since there was a large difference in the E-total value, the 3rd position of Ganodermic Acid TQ (14) was a carbonyl group, which led to a difference with tubulin via the 15th acetoxy group. The interaction may be stable.

  Therefore, Ganoderma tripenoids (1), (3)-(8), (10), (12)-(14), (16)-(24), (28), (34) and tubulin From the above E-total value indicating the stability of the interaction, it is possible to infer each effect of tumor suppression ability and antifungal performance when used as an active ingredient for various uses such as foods, cosmetics, and antifungal agents. .

  Of these, ganoderic acid TQ (compound (14)) particularly strongly interacts with tubulin, and as a result, has high tubulin polymerization promoting activity and can be said to be excellent as a tubulin polymerization inhibitor.

  Moreover, about each compound (1), (3)-(8), (10), (12)-(14), (16)-(24), (28), (34), Etotal value and (beta) tube Since the binding mode with phosphorus is different, it is not only useful as a compound (research reagent) for searching for a triterpenoid having a higher tubulin polymerization inhibitory activity with respect to triterpenoids derived from Ganoderma lucidum, but also which signal for each compound It is also useful as a compound (research reagent) for analyzing whether apoptosis occurs in cells via the transmission system.

[Test Example 4]
(Effects of Ganoderma Chloroform Extract and Hot Water Extract on Degree of Tubulin Polymerization)
To examine whether Ganoderma lingzhi extract also has a tubulin polymerization-promoting effect, two types of Ganoderma extract, namely Ganoderma hot water extract and Ganoderma chloroform extract, were used for tubulin polymerization. A degree measurement test was conducted.

(experimental method)
-Ganoderma hot water extract 121 ° C 20 min. Prepared by autoclave (same conditions as normal autoclave sterilization). The above reishi was purchased from Toyo Tanshien Co., Ltd.
-Ganoderma Chloroform Extract Room temperature 3 days Prepared by shaking extraction For each extract of Ganoderma hot water extract and Ganoderma chloroform extract, use paclitaxel as positive control and DMSO as negative control Tubulin polymerization activity (ΔRFU average) was evaluated by “Tubulin Polymerization Assay Kit” (Cytoskelton, Inc.). After adding buffer and tubulin to the 96-well plate, the final concentration in the system was 2.73 μM for paclitaxel (taxol) (positive control), and 18.2 μM for Ganoderma hot water extract and Ganoderma chloroform extract. The above extract and the like were added as samples. For the negative control, no compound or the like was added. All the samples were dissolved in a mixed solution of pure water: DMSO = 9: 1. The buffer is a fluorescent substance DAPI (excitation wavelength (Ex): 360 nm, fluorescence wavelength (Em): 420 nm) that increases the excitation intensity at 360 nm and the fluorescence intensity at 420 nm by binding to microtubules, which are tubulin polymers. In this Test Example 4, the degree of polymerization of tubulin in each well was measured by measuring the fluorescence intensity. The fluorescence intensity was measured every minute from 0 minutes to 60 minutes after the start of measurement, and was measured 61 times in total. The ΔRUF average indicates an average value of RFU per unit time (1 minute).

  (Results and discussion)

Table 6 shows the average value (ΔRFU average) in 61 measurements of the fluorescence intensity (RFU) obtained for the hot water extract and the chloroform extract of Ganoderma lingzhi . In the Ganoderma chlorophyll extract, fluorescence intensity higher than that of Negative control, that is, tubulin polymerization promoting activity was observed. In addition, with respect to the chloroform extract, it has been confirmed that tubulin polymerization promoting activity can be obtained at 0.136 mg / mL or more, but when the concentration of the chloroform extract is decreased, it is the same as the control. Up to the concentration just before the ΔRFU average value, the tubulin polymerization activity promoting effect can be expected.

On the other hand, as shown in Table 6 above, the Ganoderma hot water extract had a lower ΔRFU average value than the negative control, and showed an activity of inhibiting tubulin polymerization.
Referring to FIG. 7, when the concentration of the hot water extract is set to 1.820 mg / mL, it can be understood that the ΔRFU average is a lower value (a value about 10 to 20 lower).

  Accordingly, the tubulin polymerization inhibitory activity of the hot water extract is at least 455 mg / mL to 1.820 mg / mL, and at least a tubulin polymerization inhibitory effect is obtained, and the same effect can be expected up to the upper limit of dissolution.

[Test Example 5]
(Confirmation of the presence of Ganoderma triterpenoids in Ganoderma chloroform extract)
LCMS analysis was performed on the chloroform extract of Ganoderma lingzhi in Test Example 4 and the triterpenoid preparation of the ganoderma lucidum to verify whether the chloroform extract contained ganoderma triterpenoids.

Samples (A) to (E) for LCMS analysis were prepared by dissolving the chloroform extract and standard mixture (standards 1 to 4) in 100% methanol at the following concentrations.
(A) Chloroform extract of Test Example 3; 2 mg / mL
(B) Standard 1: Ganodermic Acid A (0.25 mg / mL) + Ganoderic Acid B (0.25 mg / mL) + Ganorside Acid A (0.25 mg / mL) + Ganodeliol F (0.25 mg / mL)
(C) Standard 2: Ganodermic Acid A (0.25 mg / mL) + Ganodermic Acid DM (0.25 mg / mL) + Ganodermanone Triol (0.17 mg / mL) + Lucaldehyde A (0.17 mg / mL) mL)
(D) Standard 3: Ganodermic Acid A (0.25 mg / mL) + Ganoderic Acid C1 (0.25 mg / mL) + Lucaldehyde B (0.25 mg / mL) + Ganoderic Acid H (0.25 mg / mL) mL)
(E) Standard 4: Ganodermic Acid A (0.25 mg / mL) + Ganoderic Acid TQ (0.27 mg / mL) + Ganoderol A (0.18 mg / mL) + Ganodermanonediol (0.14 mg) / ML)
Each sample (A)-(E) was used for "LCMS-IT-TOF" (LCMS-2020, Shimadzu Corporation) under the following conditions. Since Ganoderma triterpenoids generally have a maximum absorption wavelength at 252 nm, the detection wavelength was set to 252 nm.

(conditions)
Column used: “CAPCELL PACK C18” manufactured by SHISEIDO (column inner diameter: 1 mm, column length: 150 mm, average particle diameter of each particle of the packing material: 5 μm)
・ Injection volume: 5μL
・ Flow rate: 0.1 mL / min
LC detection wavelength: 252 nm
-Column temperature: 30 ° C
-Eluent composition (gradient):
Line A: 0.1% AcOH-Water,
Line B: 0,1% AcOH-AcCN, 25-35% B in 0-35 min, 35-45% B in 35-45 min, 45-100% in 45-145 min, 100% B in 145-160 min

(Results and discussion)
10 to 18 show the MS spectrum, MS / MS spectrum and retention time of each sample obtained from Standards 1 to 4. A chromatogram of the chloroform extract is shown in FIG. The MS spectra of the peaks indicated by numerals 1 to 3 in FIG. 19 are shown in FIGS. 20 to 22, it is estimated from the retention time and MS spectrum of each peak that peak 1 is ganodermic acid B, peak 2 is ganodermic acid A, and peak 3 is ganodermic acid DM.

  As described above, the tubulin polymerization activity regulator according to the present invention has been described in detail based on the embodiments and test examples. However, the present invention is not limited to the above-described embodiments and the like, and is described in the claims. Design changes are allowed without departing from the scope of the present invention.

Claims (12)

A tubulin polymerization activity regulator characterized by containing as an active ingredient an ingredient obtained by extracting water from a ganoderma lingzhi with a solvent containing water (aqueous solvent). . Tubulin polymerization characterized by containing tripenoids obtained by extraction from Ganoderma lingzhi with a solvent containing an organic solvent (organic solvent) as an active ingredient and promoting tubulin polymerization Activity regulator. The tripenoids are
Ganoderic acid Z, Ganoderic acid N, Ganoderic acid S, Ganoderic acid F, Gnoderic acid SZ, Ganoderic acid SZ, Ganodermic acid Z Derrick acid LM2 (Ganoderic acid LM2), ganoderic acid AM1 (Ganoderic acid AM1), ganoderic acid B (Ganoderic acid B), ganoderic acid D (Ganoderic acid D), ganoderic acid Y (Ganoderic acid Y), ganot Derrick acid TN (Ganoderic acid TN), Ganodermanic acid TQ (Ganoderic acid TQ), Ganolucidic acid A, Ganodermanontriol, Ganodermanondiol, Ganodermanondiol A), Ganoderol B (Ganoderol B), Ganoderol F (Ganoder iol F), Lucialdehyde A, Lucialdehyde B, Ganoderic acid C1, Ganoderic acid C2, Ganoderic acid C6, Ganoderic acid H (Ganoderic acid H), Ganoderic acid K (Ganoderic acid K), Ganoderic acid TR (Ganoderic acid TR), Ganoderic acid A (Ganoderenic acid A), Ganoderic acid C (Ganoderenic acid C), Ganoderic acid The compound is one or more compounds selected from the group consisting of D (Ganoderenic acid D), ganoderic acid F, and ganoderic acid H. 2. The tubulin polymerization activity regulator according to 2.   The tubulin polymerization activity regulator according to claim 1, wherein the active ingredient is a polysaccharide containing β-glucan.   The tubulin polymerization activity according to any one of claims 1 to 3, wherein the solvent is water in the case of an aqueous solvent, and is chloroform, alcohol or hydrous alcohol in the case of an organic solvent. Regulator.   A food, cosmetic or pharmaceutical product for treating, preventing or alleviating a disease associated with tubulin polymerization, comprising the tubulin polymerization activity regulator according to any one of claims 1 to 5 as an active ingredient.   The research reagent which uses the tubulin polymerization activity regulator as described in any one of Claims 1-5 as an active ingredient.   The food, cosmetic or pharmaceutical product according to claim 6, characterized in that the disease is a neoplastic disease, psoriasis or familial geothermal fever.   The antifungal agent which uses the tubulin polymerization activity regulator as described in any one of Claims 1-5 as an active ingredient.   A bittering agent comprising the tubulin polymerization activity regulator according to any one of claims 1 to 5 as an active ingredient. An action of inhibiting tubulin polymerization, comprising a step of extracting a component having an inhibitory action on tubulin polymerization from a ganoderma lingzhi using a solvent containing water (aqueous solvent). A method for producing a tubulin polymerization activity regulator. Promoting tubulin polymerization activity, characterized by including a step of extracting a component having a tubulin polymerization promoting action from Ganoderma lingzhi using a solvent containing an organic solvent (organic solvent). A method for producing a tubulin polymerization activity regulator having an action to act.